In situ Endothelialization: Bioengineering Considerations to Translation

Improving patency rates of current cardiovascular implants remains a major challenge. It is widely accepted that regeneration of a healthy endothelium layer on biomaterials could yield the perfect blood‐contacting surface. Earlier efforts in pre‐seeding endothelial cells in vitro demonstrated success in enhancing patency, but translation to the clinic is largely hampered due to its impracticality. In situ endothelialization, which aims to create biomaterial surfaces capable of self‐endothelializing upon implantation, appears to be an extremely promising solution, particularly with the utilization of endothelial progenitor cells (EPCs). Nevertheless, controlling cell behavior in situ using immobilized biomolecules or physical patterning can be complex, thus warranting careful consideration. This review aims to provide valuable insight into the rationale and recent developments in biomaterial strategies to enhance in situ endothelialization. In particular, a discussion on the important bio‐/nanoengineering considerations and lessons learnt from clinical trials are presented to aid the future translation of this exciting paradigm.     

Chemical activation and changes in surface morphology of poly(ε-caprolactone) modulate VEGF responsiveness of human endothelial cells

The high degree of clinical routine in percutaneous transluminal coronary angioplasty (PTCA) with and without stenting has not changed the fact that a large number of coronary heart disease patients are still affected by post-operative complications such as restenosis and thrombosis. Because re-endothelialization is the crucial aspect of wound healing after cardiovascular implant surgery, there is a need for modern biomaterials to aid endothelial cells in their adhesion and functional recovery post-stenting. This study systematically examines the potential of numerous chemical polymer modifications with regard to endothelialization. Poly(ε-caprolactone) (PCL) and its chemically activated forms are investigated in detail, as well as the impact of polymer surface morphology and precoating with matrix protein. Human umbilical vein endothelial cells (HUVECs) are used to characterize endothelial cell responses in terms of in vitro viability and adhesion. As a potential component in drug eluting implants, VEGF is applied as stimulus to boost endothelial cell proliferation on the polymer. In conclusion, plasma chemical activation of PCL combined with VEGF stimulation best enhances in vitro endothelialization. Examining the impact of morphological, chemical and biological modifications of PCL, this study makes an important new contribution towards the existing body of work on polymer endothelialization.     

Selective endothelial cells adhesion to Arg-Glu-Asp-Val peptide functionalized polysaccharide multilayer

Thrombosis and in-stent restenosis are the main obstacles in the healing process after cardiovascular surgery. A promising way to achieve the healing process after percutaneous transluminal coronary angioplasty followed by stenting may rely on the rapid in situ endothelialization on the materials of implants. Several requirements are raised to achieve in situ endothelialization, of which the specifically endothelial cells (ECs) homing and the non-specific cells repulsion come first. In this work, heparin/chitosan multilayer was constructed with thromboresistant and non-specifically cell-resistant properties. The specific ECs adhesive peptide sequence Arg-Glu-Asp-Val (REDV) was then immobilized onto the pristine multilayer and the cell responses of ECs and smooth muscle cells (SMCs) were verified. It is interesting that ECs selective attachment was obtained on the REDV functionalized multilayer, whereas the multilayer maintains resisting to the SMCs. These results show that the REDV functionalized cell-resistant heparin/chitosan multilayer is a ECs selective surface, which may have great potential in cardiovascular biomaterials for in situ endothelialization.     

Surface-modified hyaluronic acid hydrogels to capture endothelial progenitor cells

A major challenge to the effective treatment of injured cardiovascular tissues is the promotion of endothelialization of damaged tissues and implanted devices. For this reason, there is a need for new biomaterials that promote endothelialization to enhance vascular repair. The goal of this work was to develop antibody-modified polysaccharide-based hydrogels that could selectively capture endothelial progenitor cells (EPCs). We showed that CD34 antibody immobilization on hyaluronic acid (HA) hydrogels provides a suitable surface to capture EPCs. The effect of CD34 antibody immobilization on EPC adhesion was found to be dependent on antibody concentration. The highest level of EPC attachment was found to be 52.2 cells per mm(2) on 1% HA gels modified with 25 μg mL(-1) antibody concentration. Macrophages did not exhibit significant attachment on these modified hydrogel surfaces compared to the EPCs, demonstrating the selectivity of the system. Hydrogels containing only HA, with or without immobilized CD34, did not allow for spreading of EPCs 48 h after cell seeding, even though the cells were adhered to the hydrogel surface. To promote spreading of EPCs, 2% (w/v) gelatin methacrylate (GelMA) containing HA hydrogels were synthesized and shown to improve cell spreading and elongation. This strategy could potentially be useful to enhance the biocompatibility of implants such as artificial heart valves or in other tissue engineering applications where formation of vascular structures is required.     

Constructing bio-layer of heparin and type IV collagen on titanium surface for improving its endothelialization and blood compatibility

The modification of cardiovascular stent surface for a better micro-environment has gradually changed to multi-molecule, multi-functional designation. In this study, heparin (Hep) and type IV collagen (IVCol) were used as the functional molecule to construct a bifunctional micro-environment of anticoagulation and promoting endothelialization on titanium (Ti). The surface characterization results (AFM, Alcian Blue 8GX Staining and fluorescence staining of IVCol) indicated that the bio-layer of Hep and IVCol were successfully fabricated on the Ti surface through electrostatic self-assembly. The APTT and platelet adhesion test demonstrated that the bionic layer possessed better blood compatibility compared with Ti surface. The adhesion, proliferation, migration and apoptosis tests of endothelial cells proved that the Hep/IVCol layer was able to enhance the endothelialization of the Ti surface. The in vivo animal implantation results manifested that the bionic surface could encourage new endothelialization. This work provides an important reference for the construction of multifunction micro-environment on the cardiovascular scaffold surface.     

Enhanced cellular mobility guided by TiO2 nanotube surfaces

The in vitro endothelial response of primary bovine aortic endothelial cells (BAECs) was investigated on a flat Ti surface vs a nanostructured TiO2 nanotube surface. The nanotopography provided nanoscale cues that facilitated cellular probing, cell sensing, and especially cell migration, where more organized actin cytoskeletal filaments formed lamellipodia and locomotive morphologies. Motile cell protrusions were able to probe down into the nanotube pores for contact stimulation, and focal adhesions were formed and disassembled readily for enhanced advancement of cellular fronts, which was not observed on a flat substrate of titanium. NOx and endothelin-1 functional assays confirmed that the nanotubes also up-regulated an antithrombic cellular state for maintaining vascular tone. The enhanced endothelial response to TiO2 nanotubes is significant for a potential modification of vascular stent surfaces in order to increase the rate and reliability of endothelialization and endothelial cell migration onto the stent for repairing arterial injury after activation.     

A biodegradable and biocompatible regular nanopattern for large-scale selective cell growth

A biodegradable substrate with a regular array of nanopillars fabricated by electron-beam lithography and hot embossing is used to address the mechanisms of nanotopographical control of cell behavior. Two different cell lines cultured on the nanopillars show striking differences in cell coverage. These changes are topography- and cell-dependent, and are not mediated by air bubbles trapped on the nanopattern. For the first time, a strong cell-selective effect of the same nanotopography has been clearly demonstrated on a large area; while fibroblast proliferation is inhibited, endothelial cell spreading is visibly enhanced. The reduced fibroblast proliferation indicates that a reduction of available surface area induced by nanotopography might be the main factor affecting cell growth on nanopatterns. The results presented herein pave the way towards the development of permanent vascular replacements, where non-adhesive, inert, surfaces will induce rapid in situ endothelialization to reduce thrombosis and occlusion.     

Ultrathin Dual‐Scale Nano‐ and Microporous Membranes for Vascular Transmigration Models

Selective cellular transmigration across the microvascular endothelium regulates innate and adaptive immune responses, stem cell localization, and cancer cell metastasis. Integration of traditional microporous membranes into microfluidic vascular models permits the rapid assay of transmigration events but suffers from poor reproduction of the cell permeable basement membrane. Current microporous membranes in these systems have large nonporous regions between micropores that inhibit cell communication and nutrient exchange on the basolateral surface reducing their physiological relevance. Here, the use of 100 nm thick continuously nanoporous silicon nitride membranes as a base substrate for lithographic fabrication of 3 µm pores is presented, resulting in a highly porous (≈30%), dual‐scale nano‐ and microporous membrane for use in an improved vascular transmigration model. Ultrathin membranes are patterned using a precision laser writer for cost‐effective, rapid micropore design iterations. The optically transparent dual‐scale membranes enable complete observation of leukocyte egress across a variety of pore densities. A maximal density of ≈14 micropores per cell is discovered beyond which cell–substrate interactions are compromised giving rise to endothelial cell losses under flow. Addition of a subluminal extracellular matrix rescues cell adhesion, allowing for the creation of shear‐primed endothelial barrier models on nearly 30% continuously porous substrates.     

Stent strut streamlining and thickness reduction promote endothelialization

Stent thrombosis (ST) carries a high risk of myocardial infarction and death. Lack of endothelial coverage is an important prognostic indicator of ST after stenting. While stent strut thickness is a critical factor in ST, a mechanistic understanding of its effect is limited and the role of haemodynamics is unclear. Endothelialization was tested using a wound-healing assay and five different stent strut models ranging in height between 50 and 150 µm for circular arc (CA) and rectangular (RT) geometries and a control without struts. Under static conditions, all stent strut surfaces were completely endothelialized. Reversing pulsatile disturbed flow caused full endothelialization, except for the stent strut surfaces of the 100 and 150 µm RT geometries, while fully antegrade pulsatile undisturbed flow with a higher mean wall shear stress caused only the control and the 50 µm CA geometries to be fully endothelialized. Modest streamlining and decrease in height of the stent struts improved endothelial coverage of the peri-strut and stent strut surfaces in a haemodynamics dependent manner. This study highlights the impact of the stent strut height (thickness) and geometry (shape) on the local haemodynamics, modulating reendothelialization after stenting, an important factor in reducing the risk of stent thrombosis.     

Thromboresistant and endothelialization effects of dopamine-mediated heparin coating on a stent material surface

Heparinization of surfaces has proven a successful strategy to prevent thrombus formation. Inspired by the composition of adhesive proteins in mussels, the authors used dopamine to immobilize heparin on a stent surface. This study aimed to assess the thromboresistant and endothelialization effects of dopamine-mediated heparin (HPM) coating on a stent material surface. The HPM was synthesized by bonding dopamine and heparin chemically. Cobalt–chromium (Co–Cr) alloy disks were first placed in the HPM solution and applied to surface stability then underwent thromboresistant tests and human umbilical vein endothelial cells (HUVEC) cytotoxicity assays. The results showed not only thromboresistant activity and a stable state of heparin on the surfaces after investigation with toluidine blue and thrombin activation assay but also proliferation of HUVEC in vitro. Studies on animals showed that the HPM-coated stent has no obvious inflammation response and increasing of restenosis rate compared to the bare metal stent (BMS) indicating good biocompatibility as well as safety in its in vivo application. Moreover, improving the endothelial cell (EC) proliferation resulted in a higher strut-covering rate (i.e., endothelialization) with shuttle-shaped EC in the HPM-coated stent group compared to that of the BMS group. These results suggest that this facile coating approach could significantly promote endothelialization and offer greater safety than the BMS for its much improved thromboresistant property. Moreover, it may offer a platform for conjugating secondary drugs such as anti-proliferative drugs.     

Endothelial cell alignment on cyclically-stretched silicone surfaces

Endothelial cells at the interface between the bloodstream and the vessel wall are continuously subjected to mechanical stimulation in vivo, and it widely recognised that such stimulation plays an important role in cardiovascular physiology. Cell deformation is induced by mechanical forces such as cyclic stretch, fluid shear stress, and transmural pressure. Although much of the work in this field has dealt with the effect of fluid shear stress, very little is known about how cyclic forces modulate and alter the morphology of single endothelial cells, and thereafter, how they effect the confluent layer of endothelial cells lining the vessel wall. The aim of this study is to investigate the response of endothelial cells when subjected to substrate deformation of similar magnitude to those experienced in vivo. Human umbilical vein endothelial cells (HUVEC) were cultured on plasma-treated silicone strips and uni-axially cyclically stretched using a custom made mechanical device. Results showed that endothelial cells subject to 10% deformation for as little as 4 h reoriented perpendicular to the stretch direction. In addition, although no integrin coating was applied to the substrate, it was found that plasma-treated silicone provided a cell adhesion substrate comparable to the commonly used collagen type I. Thus the results show that the stretch stimulus alone affects the morphology of endothelial cells. Further studies are required to establish the relative importance of substrate strain vs. fluid flow stimuli.     

Accelerated endothelialization with a polymer-free sirolimus-eluting antibody-coated stent

Drug-eluting stents have shown an impressive reduction of in-stent restenosis for many years. However, stent thrombosis due to incomplete/late endothelialization has raised major safety concerns. To overcome these problems, we developed for the first time a polymer-free sirolimus-eluting antibody-coated stent (PFSEACS) by combining polymer free and endothelial progenitor cell-capture pro-healing approaches. In the first phase, the stents were prepared by loading sirolimus on the porous outer stent surface and directly fixing the anti-CD34 antibodies without any medium carriers on the blood contacting surface. The dose and elution of sirolimus, the amount and stability of anti-CD34 antibody immobilization, and the rate of CD34+ cell capture were evaluated. In the second phase, the stents were validated in an animal model of coronary arteries in pigs. The stent was observed to start collecting endothelial progenitor cells ~2 h after stent implantation and exhibited greatly enhanced endothelialization while maintaining an excellent anti-restenosis activity comparable to the polymer-free sirolimus-eluting stents. Overall, both in vitro and in vivo evaluations indicated that novel PFSEACSs exhibited facilitated endothelialization with excellent anti-restenosis activity and thus should merit further clinical studies.     

Interactions between multiple cell types in parallel microfluidic channels: monitoring platelet adhesion to an endothelium in the presence of an anti-adhesion drug

A simple method for immobilizing endothelial cells in the channels of a microfluidic device fabricated with soft lithography is presented that requires no surface oxidation of the substrate material used in conjunction with the microfluidic device and is operable even with a reversible seal. Specifically, optimal conditions for culturing bovine pulmonary artery endothelial cells (bPAECs) to the surface of a Petri dish were investigated. The parameters investigated included fibronectin concentration, temperature, seeding density, and immobilization time. To enhance the utility of the device, all optimization studies, and studies involving platelet adhesion to the immobilized endothelium, were performed in parallel channels, thereby enabling improved throughput over a single channel device. The optimal conditions for cell immobilization included coating the Petri dish with 100 microg/mL fibronectin, a seeding cell density of 1.00 x 10(5) cells mL(-1), and an immobilization time of 90 min at 37 degrees C. The device was then employed to monitor the physical interaction (adhesion) of platelets to the immobilized endothelium in the presence of a known platelet activator (ADP) and a drug inhibitor of platelet activation. The number of platelets adhering to the endothelial cells in the channels increased from 17.0 +/- 2.3 in the absence of ADP to 63.2 +/- 2.4 in the presence of 5.00 microM ADP. Moreover, the data presented here also shows that inhibition of endothelium nitric oxide (NO) production, a recognized inhibitor of platelet adhesion to the endothelium, increased the number of platelets adhering to the surface to 35.4 +/- 1.0. In the presence of NO inhibition and 5.00 microM ADP, the affect on platelet adhesion was further increased to 127 +/- 5.2. Finally, this device was employed to investigate the effect of a drug known to inhibit platelet adhesion (clopidogrel) and, in the presence of the drug, the platelet adhesion due to activation by 5.00 microM ADP decreased to 24.0 +/- 3.8. This work is the first representation of multiple cell types physically interacting in the channels of a microfluidic device and further demonstrates the potential of these devices in the drug discovery process and drug efficacy studies.     

Surface tailoring of poly(ethylene terephthalate) via ligand-tethered comb-like PEG to enhance endothelialization

The comb-like PEG (CPEG) end-tethered with l-lysine was explored to surface modification of PET to enhance endothelialization. The hydroxyl end groups of CPEG were oxygenated into aldehyde groups. The CPEG-CHO was grafted onto the aminolysized PET. The l-lysine was then end-tethered onto surface via the residual aldehyde groups. The surface modification was confirmed by ATR-FTIR, contact angle and XPS measurements. The endothelial cell adhesion, proliferation and viability results indicated that the PET-CPEG resisted cell adhesion and growth, where as PET-CPEG-lysine promoted cell adhesion and growth. The MTT assay and total cell protein tests indicated that the endothelial cells on PET-CPEG-lysine had high viability. Cell spread uniformly and covered completely on the PET-CPEG-lysine. The CPEG end tethered with l-lysine could regulate cell adhesion and growth and enhance surface endotheliazation.     

The synergistic effect of aligned nanofibers and hyaluronic acid modification on endothelial cell behavior for vascular tissue engineering

The endothelialization of tissue-engineered vascular grafts (TEVGs) is considered to be an effective strategy to prevent the coagulation and restenosis of small-diameter vascular grafts. In this study, we fabricated well aligned nanofibrous scaffolds with PCL using a high speed rotating collector, modified those surfaces with hyaluronic acid (HA) and studied the synergistic effect of the scaffolds on the endothelial cells behavior in vitro. The well-aligned oriented architecture was observed by SEM images in the nanofibrous scaffolds. The contact angle measurements and FTIR-ATR evidenced that HA was successfully modified on the PCL nanofibrous scaffolds and hydrophilicity of the scaffolds was increased after HA coating. The results of adhesion and morphology of human umbilical vein endothelial cells (HUVECs) showed that the HA-coating aligned PCL (HA-aPCL) nanofibrous scaffolds could highly promote attachment and guide HUVECs bipolar spread with the parallel aligned nanofibers. Furthermore, HUVECs on the HA-aPCL formed a confluent monoendothelial cell layer and exhibited superior protein expression levels of von Willebrand factor (vWF). This study suggested that the combination of aligned nanostructure and HA modification was more capable of promoting the regeneration of functional endothelium for vascular tissue engineering than individual use.     

Photo-immobilized heparin micropatterns on Ti–O surface: preparation,characterization, and evaluation in vitro

In order to harmonize the functions of both anticoagulation and accelerating endothelialization simultaneously, the micropatterns were fabricated by photoimmobilizing heparin, functionalized with a photoreactive moiety, on 3-aminopropylphosphonic acid modified titanium oxide (Ti–O) substrates. The amount of heparin immobilized on the surfaces was determined using the toluidine blue assay. And the surface morphology of the patterns was examined using scanning electron microscopy and surface profiler. The platelet adhesion and endothelial cell behavior in terms of adhesion, proliferation, and orientation were investigated in vitro. It is clear that the heparin patterns can reduce the platelet adhesion, and promote endothelial cells spreading and proliferation compared to nonpatterned heparin sample. Furthermore, the microstripes with appropriate size can induce the cells to elongate and arrange along the stripe direction. This may suggest a new modification method for blood-contacting device.     

A novel polyurethane modified with biomacromolecules for small-diameter vascular graft applications

There is a growing demand for small-caliber tissue-engineered vascular grafts to replace damaged vessels. Fabricated scaffolds are unable to precisely mimic the mechanical properties of native vessels, provide long-term patency and support cell adhesion and growth, in particular support endothelialization. In this study, a new biodegradable poly(ether ester urethane) urea (PEEUU) was synthesized. The synthesized polyurethane was then functionalized by introducing free amino groups through aminolysis for further surface modification by immobilization of biomacromolecules on the surface of vascular grafts. The modified surfaces were then characterized using attenuated total reflectance-Fourier transform infrared spectroscopy, water contact angle measurement and atomic force microscopy. The mechanical properties of the fabricated scaffolds were analyzed, revealing mechanical properties close to that of the natural vessels. Surface modifications led to improved cell–scaffold interactions, showing appropriate cell attachment and function on the scaffolds. A confluent layer of endothelial cells was formed on biomacromolecule-immobilized PEEUU vascular grafts. The preliminary results of this study demonstrated that the new polyurethane modified with biomacromolecules can be considered as a candidate material for vascular tissue engineering application with capability to support endothelialization of fabricated vascular grafts.     

Hypergravity affects morphology and function in microvascular endothelial cells

Cardiovascular diseases are major health problems in astronauts and pilots. The basic problem in cardiovascular diseases is the loss of function by vascular endothelium. It has been demonstrated that changes in inertial conditions (i.e. hypo- and hypergravity) can affect both phenotypic and genotypic expression in endothelial cells. This report describes the effects observed in endothelial cells from coronary post-capillary venules after repeated exposures to hypergravity conditions, alternating with recovery periods. The results showed changes in gene expression, cell energy metabolism, morphology and cytoskeleton organization.     

Reversible Cavitation‐Induced Junctional Opening in an Artificial Endothelial Layer

Targeting pharmaceuticals through the endothelial barrier is crucial for drug delivery. In this context, cavitation‐assisted permeation shows promise for effective and reversible opening of intercellular junctions. A vessel‐on‐a‐chip is exploited to investigate and quantify the effect of ultrasound‐excited microbubbles—stable cavitation—on endothelial integrity. In the vessel‐on‐a‐chip, the endothelial cells form a complete lumen under physiological shear stress, resulting in intercellular junctions that exhibit barrier functionality. Immunofluorescence microscopy is exploited to monitor vascular integrity following vascular endothelial cadherin staining. It is shown that microbubbles amplify the ultrasound effect, leading to the formation of interendothelial gaps that cause barrier permeabilization. The total gap area significantly increases with pressure amplitude compared to the control. Gap opening is fully reversible with gap area distribution returning to the control levels 45 min after insonication. The proposed integrated platform allows for precise and repeatable in vitro measurements of cavitation‐enhanced endothelium permeability and shows potential for validating irradiation protocols for in vivo applications.     

Nitric Oxide Donors for Cardiovascular Implant Applications

In an era of increased cardiovascular disease burden in the ageing population, there is great demand for devices that come in to contact with the blood such as heart valves, stents, and bypass grafts that offer life saving treatments. Nitric oxide (NO) elution from healthy endothelial tissue that lines the vessels maintains haemostasis throughout the vasculature. Surgical devices that release NO are desirable treatment options and N‐diazeniumdiolates and S‐nitrosothiols are recognized as preferred donor molecules. There is a keen interest to investigate newer methods by which NO donors can be retained within biomaterials so that their release and kinetic profiles can be optimized. A range of polymeric scaffolds incorporating microparticles and nanomaterials are presenting solutions to current challenges, and have been investigated in a range of clinical applications. This review outlines the application of NO donors for cardiovascular therapy using biomaterials that release NO locally to prevent thrombosis and intimal hyperplasia (IH) and enhance endothelialization in the fabrication of next generation cardiovascular device technology.